Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).     

Heterogeneous diffusion in aerobic granular sludge

Aerobic granular sludge (AGS) technology allows simultaneous nitrogen, phosphorus, and carbon removal in compact wastewater treatment processes. To operate, design, and model AGS reactors, it is essential to properly understand the diffusive transport within the granules. In this study, diffusive mass transfer within full-scale and lab-scale AGS was characterized with nuclear magnetic resonance (NMR) methods. Self-diffusion coefficients of water inside the granules were determined with pulsed-field gradient NMR, while the granule structure was visualized with NMR imaging. A reaction-diffusion granule-scale model was set up to evaluate the impact of heterogeneous diffusion on granule performance. The self-diffusion coefficient of water in AGS was ∼70% of the self-diffusion coefficient of free water. There was no significant difference between self-diffusion in AGS from full-scale treatment plants and from lab-scale reactors. The results of the model showed that diffusional heterogeneity did not lead to a major change of flux into the granule (<1%). This study shows that differences between granular sludges and heterogeneity within granules have little impact on the kinetic properties of AGS. Thus, a relatively simple approach is sufficient to describe mass transport by diffusion into the granules.     

Search for α3β2/3γ2 subtype selective ligands that are stable on human liver microsomes

Selective modulation of specific benzodiazepine receptor (BzR) gamma amino butyric acid-A (GABA_A) receptor ion channels has been identified as an important method for separating out the variety of pharmacological effects elicited by BzR-related drugs. Importantly, it has been demonstrated that both α2β_(2/3)γ2 (α2BzR) and α3BzR (and/or α2/α3) BzR subtype selective ligands exhibit anxiolytic effects with little or no sedation. Previously we have identified several such ligands; however, three of our parent ligands exhibited significant metabolic liability in rodents in the form of a labile ester group. Here eight analogs are reported which were designed to circumvent this liability by utilizing a rational replacement of the ester moiety based on medicinal chemistry precedents. In a metabolic stability study using human liver microsomes, four compounds were found to undergo slower metabolic transformation, as compared to their corresponding ester analogs. These compounds were also evaluated in in vitro efficacy assays. Additionally, bioisostere 11 was evaluated in a rodent model of anxiety. It exhibited anxiolytic activity at doses of 10 and 100 mg/kg and was devoid of sedative properties.     

Anti-tumoral,anti-angiogenic and anti-metastatic efficacy of a tetravalent bispecific antibody (TAvi6) targeting VEGF-A and angiopoietin-2

Vascular endothelial growth factor (VEGF)-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) is a key regulator of blood vessel remodeling and maturation. In tumors, Ang-2 is up-regulated and an unfavorable prognostic factor. Recent data demonstrated that Ang-2 inhibition mediates anti-tumoral effects. We generated a tetravalent bispecific antibody (Ang-2-VEGF-TAvi6) targeting VEGF-A with 2 arms based on bevacizumab (Avastin®), and targeting Ang-2 with 2 arms based on a novel anti-Ang-2 antibody (LC06). The two Ang-2-targeting single-chain variable fragments are disulfide-stabilized and fused to the C-terminus of the heavy chain of bevacizumab. Treatment with Ang-2-VEGF-A-TAvi6 led to a complete abrogation of angiogenesis in the cornea micropocket assay. Metastatic spread and tumor growth of subcutaneous, orthotopic and anti-VEGF-A resistant tumors were also efficiently inhibited. These data further establish Ang-2-VEGF bispecific antibodies as a promising anti-angiogenic, anti-metastatic and anti-tumor agent for the treatment of cancer.     

Salt‐enhanced cultivation as a morphology engineering tool for filamentous actinomycetes: Increased production of labyrinthopeptin A1 in Actinomadura namibiensis

Salt‐enhanced cultivation as a morphology engineering tool for the filamentous actinomycete Actinomadura namibiensis was evaluated in 500‐mL shaking flasks (working volume 100 mL) with the aim of increasing the concentration of the pharmaceutically interesting peptide labyrinthopeptin A1. Among the inorganic salts added to a complex production medium, the addition of (NH₄)₂SO₄ led to the highest amount of labyrinthopeptin A1 production. By using 50 mM (NH₄)₂SO₄, the labyrinthopeptin A1 concentration increased up to sevenfold compared to the non‐supplemented control, resulting in 325 mg L^?1 labyrinthopeptin A1 after 10 days of cultivation. The performance of other ammonium‐ and sulfate‐containing salts (e.g., NH₄Cl, K₂SO₄) was much lower than the performance of (NH₄)₂SO₄. A positive correlation between the uptake of glycerol as one of the main carbon sources and nongrowth‐associated labyrinthopeptin productivity was found. The change in the cell morphology of A. namibiensis in conjunction with increased osmolality by the addition of 50 mM (NH₄)₂SO₄, was quantified by image analysis. A. namibiensis always developed a heterogeneous morphology with pellets and loose mycelia present simultaneously. In contrast to the non‐supplemented control, the morphology of (NH₄)₂SO₄‐supplemented cultures was characterized by smaller and circular pellets that were more stable against disintegration in the stationary production phase.     

Role of SP65 in assembly of the Dictyostelium discoideum spore coat

Like the cyst walls of other protists, the spore coat of Dictyostelium discoideum is formed de novo to protect the enclosed dormant cell from stress. Spore coat assembly is initiated by exocytosis of protein and polysaccharide precursors at the cell surface, followed by the infusion of nascent cellulose fibrils, resulting in an asymmetrical trilaminar sandwich with cellulose filling the middle layer. A molecular complex consisting of cellulose and two proteins, SP85 and SP65, is associated with the inner and middle layers and is required for proper organization of distinct proteins in the outer layer. Here we show that, unlike SP85 and other protein precursors, which are stored in prespore vesicles, SP65 is, like cellulose, synthesized just in time. By tagging the SP65 locus with green fluorescent protein, we find that SP65 is delivered to the cell surface via largely distinct vesicles, suggesting that separate delivery of components of the cellulose-SP85-SP65 complex regulates its formation at the cell surface. In support of previous in vivo studies, recombinant SP65 and SP85 are shown to interact directly. In addition, truncation of SP65 causes a defect of the outer layer permeability barrier as seen previously for SP85 mutants. These observations suggest that assembly of the cellulose-SP85-SP65 triad at the cell surface is biosynthetically regulated both temporally and spatially and that the complex contributes an essential function to outer layer architecture and function.